The structure of Ski8p, a protein regulating mRNA degradation: Implications for WD protein structure

Abstract

Ski8p is a 44-kD protein that primarily functions in the regulation of exosome-mediated, 3'--> 5' degradation of damaged mRNA. It does so by forming a complex with two partner proteins, Ski2p and Ski3p, which complete a complex that is capable of recruiting and activating the exosome/Ski7p complex that functions in RNA degradation. Ski8p also functions in meiotic recombination in complex with Spo11 in yeast. It is one of the many hundreds of primarily eukaryotic proteins containing tandem copies of WD repeats (also known as WD40 or beta-transducin repeats), which are short ~40 amino acid motifs, often terminating in a Trp-Asp dipeptide. Genomic analyses have demonstrated that WD repeats are found in 1%-2% of proteins in a typical eukaryote, but are extremely rare in prokaryotes. Almost all structurally characterized WD-repeat proteins are composed of seven such repeats and fold into seven-bladed beta propellers. Ski8p was thought to contain five WD repeats on the basis of primary sequence analysis implying a five-bladed propeller. The 1.9 A crystal structure unexpectedly exhibits a seven-bladed propeller fold with seven structurally authentic WD repeats. Structure-based sequence alignments show additional sequence diversity in the two undetected repeats. This demonstrates that many WD repeats have not yet been identified in sequences and also raises the possibility that the seven-bladed propeller may be the predominant fold for this family of proteins.

Figure 1.

Stereo views of the C_α trace of Ski8p oriented (A) down the propeller axis (colored by temperature factor), and (B) normal to the propeller axis (β strands are colored red; loop regions are shown in blue). Molecular graphics in Figs. 1, 2 ▶, 3 ▶, and 5 ▶ were created using BOBSCRIPT (Esnouf 1997).

Figure 2.

(A) A stereo view of structural alignments of the WD repeats in Ski8p demonstrate that all are authentic. Repeats one through seven are colored red through purple, respectively. (B) Structure-based sequence alignments of the conserved regions of the WD repeats in Ski8p, consisting of strands A, B, and C. Included are those previously identified by sequence (designated WD1–WD5) and additional repeats found in the structure (S1 and S2). Larger numbers of nonhomologous residues, which are inserted into the repeat loop regions, are abbreviated with square brackets. The consensus sequence as described by Neer et al. (1994) is shown below the alignments in descending frequency.

Figure 2.

(A) A stereo view of structural alignments of the WD repeats in Ski8p demonstrate that all are authentic. Repeats one through seven are colored red through purple, respectively. (B) Structure-based sequence alignments of the conserved regions of the WD repeats in Ski8p, consisting of strands A, B, and C. Included are those previously identified by sequence (designated WD1–WD5) and additional repeats found in the structure (S1 and S2). Larger numbers of nonhomologous residues, which are inserted into the repeat loop regions, are abbreviated with square brackets. The consensus sequence as described by Neer et al. (1994) is shown below the alignments in descending frequency.

Figure 3.

A superimposition of Ski8p (blue), Gβ (green), and Tup1p (red) in an orientation similar to that shown in Figure 1A ▶ indicate that the core fold is preserved. Structural divergence is concentrated in the loops, particularly those on the top of the propeller (directed out of the page).

Figure 4.

Confirmed and potential WD repeats in other representative budding yeast proteins. (A) Repeats that had been previously located in sequences are shown in red. Additional potential repeats are shown in yellow. In the cases of Aip1p and Ski8p, these additional repeats have been confirmed by structures. (B) Sequence alignments of additional repeats described in A. Shown are the conserved regions in strands A, B, and C, and consensus sequences as described in Figure 2 ▶.

Figure 5.

Divergence and disappearance of the structural tetrad in representative Ski8p blades. (A) Blade 1 contains a nearly conventional tetrad involving hydrogen bonding between the side chains of Trp 41, Ser 31, and His 15. The aspartate completes the tetrad in the canonical structure. (B) Blade 3 has Trp 153 and Asp 145 at the expected positions, but other residues are divergent. The core of this blade is stabilized via a mixture of hydrogen bonding and hydrophobic interactions. (C) Blade 7 contains residues that are homologous in sequence to a conventional tetrad. The positions are structurally divergent, however.